PROJECT SUMMARY The human colon houses a complex community of microbes, known as the gut microbiota, which possesses unmapped metabolic capabilities. Bacterial metabolic pathways process components of diet, like amino acids, and produce an array of ill-defined metabolites. Many of the metabolites produced by this microbial ecosystem are absorbed by the human host, modified by host enzymes, and ultimately excreted by the kidneys. When the kidneys fail, these solutes accumulate and comprise a significant portion of the uremic solutes found at very high levels in the plasma of patients maintained on dialysis. These compounds can vary widely between individual patients, yet are relatively stable over time within an individual, potentially reflecting inter-individual differences in gut microbiota composition. A few of these molecules have been investigated and linked to poor health outcomes in renal patients. For most of these compounds, however, neither the biochemical pathways responsible for their formation nor their biological effects on the host have been elucidated. This application is focused on the prevalent high concentration uremic solutes derived from tyrosine, 4- ethylphenylsulfate (4-EPS) and p-cresolsulfate (PCS), as well as 4-hydroxyphenylpropionic acid sulfate, a tyrosine metabolite not associated with uremia but important in understanding the tyrosine-utilization niche within the gut ecosystem. The goals of the research are to (i) determine the genes and species within the gut microbiota responsible for production of the microbial metabolites 4-ethylphenol and p-cresol that serve as precursors to 4-EPS and PCS; (ii) elucidate the effects of these molecules on aspects of host biology relevant to uremic illness; and (iii) investigate two distinct strategies for microbiota reprogramming with a goal of lowering uremic solute levels in a host. Aim 1 employs two approaches to predict microbial metabolic pathways, one using a computational/machine learning approach and a second method using comparative genomics combined with bacterial metabolomic phenotyping. Gene predictions will be genetically validated using gene deletion or heterologous expression. In Aim 2, gnotobiotic mice are used as a platform to investigate the conversion of microbial metabolites into circulating solutes, and how solute levels are affected by diet and other members of the microbiota. Isotopically labeled amino acids are used to trace dietary substrates to uremic solute products. Aim 3 leverages gnotobiotic mice colonized by WT versus mutant bacteria, which differ in the presence or absence of 4-EPS or PCS, to examine the effect of the metabolite on host biology. Changes in arterial thrombosis and cognitive function relevant to uremic illness will be assessed. The focus of Aim 4 is to reprogram the microbiota to reduce production of harmful uremic solutes. Single strain targeted reprogramming or complex consortium-based microbiota reconstitution using a diverse array of culturable bacteria will be tested as complementary strategies for lowering uremic solute levels in mice. Dietary modifications or antibiotic-based ablation of the microbiota will be used to augment the reprogramming therapies, respectively.